Broadly speaking, there are two alternative approaches to the manipulation
of genetic expression through transformation:

Transformation with novel genes to confer a new trait, or with modified
genes to enable more effective performance of an existing function.
These traits, e.g. herbicide tolerance, insect resistance and cold
tolerance, are discussed below.

Transformation with genes which lead to the blocking of protein
synthesis, through either antisense or ribozyme technology. With the
antisense approach, a gene is transcribed in reverse direction, and an
mRNA is produced that is complementary to, and thus able to bind, the
normal message - blocking protein synthesis. Objectives can include
reducing production of unwanted products by targetting particular
enzymatic steps in a pathway, or viral resistance by targetting viral
gene expression and/or replication. The expression of antisense RNA is
a powerful method for the down-regulation of expression of the
corresponding sense RNA (Harms 1992), but requires delivery of a large
excess of antisense over sense mRNA because the antisense/mRNA
hybrids are kinetically constrained (Ratner 1989). With the ribozyme
approach, the RNA molecules that are synthesized have the capacity
to cleave mRNA's of target genes - essential genes of insects or
perhaps viruses. Being catalytic molecules, ribozymes are likely to be
more effective at destroying the translational capacity of mRNAs than
antisense transcripts (Harms 1992).

Transformation methods were described in detail by Potrykus (1990).
Included in these descriptions are some which are unproven or of limited
use, e.g. the use of virus vectors, incubation of tissues in DNA solutions,
pollen transformation, microlasers, electrophoresis, and macro and
microinjection. Widely proven techniques for the production of transgenic
plants are Agrobacterium mediated transfer, and the direct gene transfer
methods - bombardment with DNA-coated microprojectiles, and
electroporation or polyethylene glycol (PEG) treatment of protoplasts (Birch
1993).

Agrobacterium is a soil pathogen that naturally parasitizes plants by
inserting a part of its own DNA into the host's genome. A. rhizogenes and
A. tumefaciens transfer a portion of one of their large plasmids (termed Ri
and Ti respectively) to the chromosomal DNA of a plant when the
bacterium infects the wound. The transferred DNA (T-DNA) encodes
functions that induce root formation in the former case and tumour
formation in the latter. These natural transformation systems have been
used to introduce non-T-DNA genes into plants, by inserting the foreign
gene into the T-DNA itself, or by using a binary vector system, where the
foreign gene is carried by an artificial T-DNA on a second plasmid or in a
second bacterium (Tepfer 1990). One of the most important findings was
that disarmed T-DNA (i.e. without functional oncogenes) can be transferred
and integrated into the plant genome, allowing regeneration of transgenic
plants (Binns 1990). Uncertainties remain concerning how the T- DNA
crosses the cell wall, and how it becomes integrated. Under optimal
conditions, transformation efficiency is very high and genes are stably
integrated, but variation in host sensitivity has been a limitation.
Agrobacterium infects nearly all dicots, and some conifers, but generally
not monocots (Grimsley 1990, Laimer da Camara Machado 1992, Potrykus
1990). Even within a species, variation in sensitivity to infection can be
substantial (Potrykus 1990, Binns 1990, Bergmann & Stomp 1992). In a
study with Pinus radiata, host genotypes differed significantly in
susceptibility to infection, although bacterial strain and bacterial strain ×
host genotype effects were not significant. A conclusion of this study was
that a major component of the host-pathogen interaction may be the
genetic and environmental factors controlling host plant cell division at the
time of inoculation (Bergmann & Stomp 1992).

Protoplast methods basically involve removal of cell walls to yield a
protoplast suspension, and then the use of chemical (PEG) or electrical
(“electroporation”) treatments to increase membrane permeability to DNA
transfer (Lindsay & Jones 1990). While the use of protoplasts eliminates
a major barrier to the passage of DNA (the cell wall), regeneration of plants
from protoplasts is difficult for many species.

Biolistic methods involve the acceleration of small metal particles coated
with DNA to deliver DNA into plant tissues, through the cell wall.
Acceleration of gold or tungsten particles is achieved by an explosive
charge or by gas flow, the latter reportedly cheaper and more efficient
(Finer et al. 1992, Takeuchi et al. 1992). Helium gas is used because of
low mass and high diffusivity. The biolistic approach is relatively simple
technically, avoids the requirement for removal of the cell wall, and is good
for producing transient expression signals. The transition from transient to
stable integrative transformation, however, is very inefficient.
Transformation of chloroplasts has been achieved by particle bombardment
(Potrykus 1990).

Comparative features of the major transformation methods were
tabulated by Birch (1993):

Table 1. Features of Methods for Transgenci Plant Production (Birch 1993)

Promoters are DNA sequences which initiate the transcription of adjacent
gene coding sequences and which are modulated by additional adjacent
DNA sequences known as enhancers (Strauss et al. 1991, Whetton &
Sederoff 1991). Most commonly used experimentally is the cauliflower
mosaic virus 35S promoter, a promoter active in most stages of
development and in most plant tissues. Effective applications of genetic
engineering, however, are frequently likely to be dependent on the use of
promoters specific to particular tissues or developmental events. It is
desirable, for example, for insect resistance to be expressed only after
insects begin to feed and in the tissues under attack - requiring the splicing
to the resistance gene of a combination of suitable promoters. The
approach to finding such promoters is to identify any gene expression with
the required tissue or developmental specificity, and then to isolate the
regulatory elements and test for specificity. Many promoters directing
specific patterns of gene expression are known:

This field is moving rapidly, but much remains to be done. An important
question is the extent to which promoters will fuction across wide
taxonomic boundaries. Some results are encouraging - in particular the
demonstrated evolutionary conservation of tissue specific expression of
ribulose biphosphate carboxylase activity between conifers and
angiosperms (Campbell & Neale 1992).

Both experimentally and for the practical application of genetic engineering,
a method of verifying transformation or selecting transformed from
untransformed tissues is necessary. For experimental purposes, success of
Agrobacterium mediated transfer has often been judged by expression of
bacterial genes transferred - tumour formation, formation of hairy roots and
production of opines (characteristic amino acids). More generally though,
the addition of marker genes to the constructs is required. Marker genes
employed include:

B-glucuronidase (GUS) - determined using a characteristic but lethal
assay

Herbicide or antibiotic assays which kill or suppress non-transformed
cells, while allowing proliferation of transformed tissues, are attractive, but
sensitivity of tissues to the selecting agent is critical. A high incidence of
untransformed “escapes” has been demonstrated in some studies (Ellis,
McCabe et al. 1992).

The insecticidal properties of crystal proteins produced by this bacterium
have long been known, B.t. based bioinsecticides have been used since
1935, and the current annual market is estimated at $US 100 million (van
Montagu 1992a). 22 different crystal proteins and their corresponding
genes have been identified. Based on structural relationship and spectrum
of activity, these are grouped into Lepidoptera-specific, Lepidoptera and
Diptera specific, Diptera specific, and Coleoptera specific (van Montagu
1992a). Specificity of the proteins is determined mainly by their binding to
specific receptors located in the membrane of the midgut of larvae.
Insecticidal activity involves two steps: - binding to the receptor and then
toxicity caused by integration into the membrane and creation of a pore
(Frutos unpublished). One of the first successful applications of plant
transformation technology for crop improvement, insect resistant tobacco,
tomato, potato, and cotton engineered with four different crystal genes
has been reported. Compared to other genes transferred to plants,
insecticidal crystal protein genes are weakly expressed in transgenic plants
(van Montagu 1992a). B.t. based insecticides have been used on some
forest crops, and development of trees that can produce the toxin is
underway in a number of species (Strauss et al. 1991). Development of
resistance to B.t. toxins, e.g. in Lepidoptera, has been reported. Such
resistance is not due to an active process like destruction of the protein,
but to a “passive” process - individuals selected for resistance are those
with a modified receptor, unable to be recognized by the activated toxin
(Frutos, unpublished).

Several chitinase genes have been cloned and some transferred into
tobacco, but effects on insect pests are yet to be reported (Strauss et al.
1991). Plant secondary products are a major means by which trees protect
themselves against insects, and potential exists for importing encoding
genes from suitable sources - e.g. the neem tree. Many natural resistance
mechanisms in trees, however, are under polygenic control, are of
unknown molecular basis, and are not likely to be amenable to genetic
engineering in the near future (Strauss et al. 1991).

Stable resistance to insects is best achieved by the incorporation of
multiple resistance genes - preferably including more than one class.

In most cases, resistance has been engineered by incorporating into the
host genome a gene from the virus, most commonly, although not
exclusively, the virus coat protein gene (Harms 1992, Hoekema et al.
1989, Beachy et al. 1992, Zaitlin 1991). Expression of the coat protein
gene in transgenic plant cells interferes with the uncoating of the incoming
virus. Resistance is frequently conferred also to other related viruses, and
shows considerable resemblance to the phenomenon of virus
cross-protection (Harms 1992). It has been suggested that genes leading
to the accumulation of several non-functional viral proteins, including
capsid proteins, insect transmission genes, proteases, and movement
proteins, might also provide resistance against infection, or transmission
between plants or between cells (Beachy et al. 1992). In an alternative
approach, tobacco resistant to tomato golden mosaic virus was obtained
by transformation with an antisense DNA sequence of a gene encoding a
protein absolutely required for viral replication (Bejarano 1991).

Coat protein mediated resistance has been demonstrated in field trials,
now aged up to four years, in several countries, and it is anticipated that
virus resistant transgenic agricultural plants will be released for commercial
use in the mid-1990s (Beachy et al. 1992).

Few reports exist of successful engineering of bacterial or fungal
resistance. Tobacco transformed with genes for thionins (polypeptides that
are toxic to bacterial and fungal pathogens) showed resistance when
challenged with bacterial pathogens (Carmona 1991). The Septoria leaf
spot and stem canker pathogen of poplar was found to produce
proteinases which are inhibited by purified proteinase inhibitor II protein,
and studies are underway with transgenic plants to determine the effect
of the proteinase inhibitor gene on infection and development (McNabb et
al. 1990). An alternative approach is to seek detoxification enzymes in
pathogens producing phytotoxins, and to transfer the encoding genes to
plants in which resistance is desired - this has been done experimentally
with Pseudomonas syringae and tobacco (Harms 1992).

No transgenic plants resistant to nematodes have been reported to date,
but a number of lines of research are being pursued (De Waele 1992):

Lytic enzymes and toxic proteins to kill nematodes. Lytic enzymes
include chitinases and collagenases, and toxins include the B. t.
toxin. Isolates of the latter with activity against nematodes have
been identified.

Cloning of genes known to confer resistance, e.g. the Mi gene from
Lycopersicon peruvianum which is effective against almost all
Meloidogyne species attacking tomato.

Interference in the nematode-plant interaction, e.g. using proteins
blocking recognition.

Usually a genetically simple trait with a clear unambiguous phenotype,
herbicide tolerance is particularly amenable to genetic manipulation (Chaleff
1986b). Many herbicides act by inhibiting important enzymes. Three
resistance strategies are available:

Detoxification.
Glutathione-S transferases, for example, catalyze the conjugation of
glutathione to an electrophilic centre of hydrophobic herbicides and
thereby confer resistance to herbicides of the s-triazine type. Some
crop species have these enzymes naturally - e.g. maize and sorghum
can detoxify atrazine (Schulz et al. 1990). Transformation with the
bacterial Bar gene encoding phosphinothricin acetyl transferase
(PAT), which acetylates phosphinothricin, has given rise to
phosphinothricin-resistant transgenic plants for both tobacco (Schulz
et al. 1990) and wheat (Vasil et al. 1992). A possible source of
genes which code enzymes that metabolize nonselective herbicides
is the wide spectrum of microbial organisms which detoxify herbicides
in the soil (Schulz et al. 1990).

Overproduction of the enzyme.
Glyphosate is a potent competitive inhibitor of the enzyme
5-enol-pyruvylshikimicacid 3- phosphate (EPSP) synthase, which is
involved in the biosynthesis of aromatic amino acids. The use of EPSP
synthase in combination with a powerful promoter, resulting in
overexpression, has resulted in the production of glyphosate resistant
petunia plants (Schulz et al. 1990). The herbicide phosphinothricin is
a strong inhibitor of glutamine synthetase. Fusing of the alfalfa
glutamine synthetase gene to the 35S promoter and transferring to
tobacco resulted in over expression of the gene and a large increase
in resistance to the herbicide in the transgenic plants (Schulz et al.
1990).

Mutant enzymes.
The aroA gene which encodes EPSP synthase has been isolated and
sequenced from several organisms. A mutated bacterial aroA gene
encoding a resistant EPSP synthase has been used to produce
glyphosate resistant transgenic petunia plants (Schulz et al. 1990).
The herbicidal effect of imidazolinones and the sultonylureas is in
both cases due to inhibition of the enzyme acetolactate synthase
(ALS), which is the first enzyme in the biosynthesis of the branched
chain amino acids. Mutated forms of the enzyme, conferring
herbicide resistance, are known (Schulz et al. 1990).

A gene coding for the winter flounder antifreeze protein was introduced
into corn protoplasts by electroporation, and expression subsequently
observed in the protoplasts (Cutler 1991). Expression was reported also in
tobacco plants transformed with a gene construct encoding an antifreeze
protein of ocean pout (Kenward 1992). A small proportion of antifreeze
protein (10–20 ng in 120 ug of protein analysed) was sufficient to displace
the survival curve for transgenic plantlets by approx. 1°C in the direction
of increased freeze tolerance. Antifreeze protein has been inserted also into
potato, but cold tolerance of the plants not tested (Garcia et al. 1992).

With the exception of cell wall proteins, wood components are products
of reactions catalyzed by many enzymes, and genetic manipulation is
therefore a matter of manipulation of genes which encode key enzymes in
various biosynthetic pathways (Whetton & Sederoff 1991). The pathway
leading to lignin formation is well characterized for many species, and
many projects aimed at lignin modification are underway. General
strategies involved are:

Reduction of lignin production. Enzyme regulation of the aromatic
amino acid, phenyl-propanoid and monolignol pathways is under
study in many laboratories, and enzymes within these pathways are
logical targets for genetic engineering. Useful constructs for lignin
reduction include those leading to overexpression of gene products
intended to scavenge extracellular free radicals, and those causing
underexpressing of gene products intended to polymerize monolignols
(Dean & Eriksson 1992). Many studies concern genes encoding the
enzymes cinnamyl alcohol dehydrogenase (CAD), the last step in the
synthesis of monolignol precursors, and phenylalanine ammonia lyase
(PAL) (Harry, Strauss & Sederoff 1991, Whetton & Sederoff 1991,
Feuillet et al. 1992, O'Malley et al. 1992). These enzymes are
encoded by single genes in Pinus, and by multigene families in
angiosperms (Harry, Strauss & Sederoff 1991). The cDNA
corresponding to eucalypt CAD has been cloned (Feuillet et al. 1992).
Underexpression of lignin associated enzymes using antisense
technology has been accomplished with CAD and a lignin-associated
peroxidase (Dean & Eriksson 1992). Other target enzymes include:

cinnamoyl-CoA reductase (CCR), the first step in the
channeling of cinnamic acid derivatives into the monolignol
biosynthetic pathway. The enzyme has been purified and a
cDNA clone is being produced using sequence data (Campbell
& Boudet 1992).

O-Methyltransferase (OMT), which plays an important role in
the synthesis of lignin precursors by catalysing the
O-methylation of o-diphenolic substrates such as caffeic acid
(Van Doorsselaere et al. 1992). Lignin content is being
assessed in poplar plants transformed with both sense and
antisense fragments of an OMT gene (Cornu et al. 1992).

Coumarate CoA ligase (CCL), an enzyme which catalyzes a
reaction from the middle of the lignin biosynthetic pathway.
This enzyme has been purified from loblolly pine and the gene
is being characterized (Voo et al. 1992).

Modification of lignin composition. This includes the modification of
softwood lignin to make it more readily hydrolyzed, by incorporation
of the hardwood genes that produce syringyl lignin. In the simplest
case, genes for only two enzymes would be needed; but many
gymnosperm enzymes do not use angiosperm substrates readily, so
that as many as seven hardwood enzymes might be required
(Whetton & Sederoff 1991). Work is proceeding to introduce an
angiosperm methyl transferase into a gymnosperm to give greater
quantities of sinapyl alcohol (Dean & Eriksson 1992).

The use of xylem specific promoters will be important in the alteration
of lignin quantity and content, since interference with lignification in
non-vascular tissues, which might play a role in resistance to pests, should
be avoided (Whetton & Sederoff 1991).

By contrast with the lignin biosynthetic pathway, those for cellulose,
hemicellulose and extractives remain poorly understood (Whetton &
Sederoff 1991). The generally poor understanding of the genetic basis of
most wood properties is a major constraint to genetic engineering of these
traits.

Cytoplasmic male sterility (CMS) has been reported in over 140 higher
plant species. The commercial use of CMS lines as female parents was
made possible by the discovery of a specific, dominant, nuclear restorer of
fertility genes (Leaver 1992). Nuclear male sterility has been engineered by
targeting expression of ribonucleases such as barnase, from Bacillus
amyloliquefaciens, specifically to the tapetum of immature anthers, leading
to tapetal degeneration and the arrest of microspore development
(Leemans 1992). Genes which can restore fertility have been constructed
by linking the tapetal promoter to barstar, the intracellular inhibitor of
barnase. This hybrid system has been successfully introduced in oilseed
rape, cauliflower, chicory, lettuce, tomato, cotton and corn (Leemans
1992).

Projects aimed at genetically engineering sterile trees of Pinus, Eucalyptus
and Populus are underway (Strauss et al. 1992 unpub.).
Two approaches are being attempted in this work:

Isolation as cDNA clones of genes expressed only during early stages
of differentiation of male and female reproductive buds. Reproductive
tissue specific genes from herbaceous species are also being used to
screen pine cDNA libraries.

Isolation of tree homologues to floral homeotic genes identified in
Arabidopsis and Antirrhinum. Such genes and their promoters should
allow the construction of novel genes which cause sterility when
inserted into trees.

The most common form of self-incompatibility in angiosperms is the single
locus multi-allelic gametophytic system (S-locus). Some gene transfer work
has been done, but the use of genetic engineering to transfer naturally
occurring self-incompatibility systems or parts thereof to functional
heterologous combinations remains a distant prospect (Olesen et al. 1992).
The sensitivity of the S-locus to its genetic background is likely to be a
constraint. Even less is known about the genetic control of cross
incompatibility. A change in activity of adenylate cyclase has been reported
in association with compatible vs incompatible pollinations in interspecific
crosses in poplars, but the molecular basis of the phenomenon remains
obscure (Olesen et al. 1992).

The expression of antisense RNA to ACC synthase in tomato fruits inhibits
ethylene production, and ripening and softening of the fruits, an effect
which can be reversed by treatment with exogenous ethylene (Theologis
1992). It has been suggested that this may offer a general method for
preventing senescence in a variety of fruits and vegetables.

Enhanced rooting of cuttings of Eucalyptus grandis, E. dunnii, E. nitens
(Haigh 1992), E. gunnii, Salix alba, Allocasuarina verticillata and some fruit
tree species (Chandler et al. 1993) has been achieved by transformation
with the natural root promoting gene from Agrobacterium rhizogenes. This
can be achieved simply by dipping the bottom of the cutting into a culture
of the bacterium. Although certain strains of the bacterium were found to
be specific to individual eucalypt clones, one strain was suitable for a wide
variety of clones (Haigh 1992). Plants are chimaeric, aerial growth is
non-transgenic, and there is no risk therefore of release of bacterial genes
through pollen or seed dispersal.

Symbiotic studies have targeted protein formation and gene expression
specific to infected tissues, during the process of signalling between the
symbionts, during the early stages of nodule formation, and in the
assimilation of fixed nitrogen during later stages of nodule ontology
(Graham 1992). Studies underway for Phaseolus, soybean, alfalfa and
other crops have resulted in the identification of some proteins and the
characterisation of a few genes. Attempts to select for enhanced levels of
enzymes involved, e.g. PEP carboxylase, glutamine synthase and
leghaemoglobin, however, did not result in improved nitrogen fixation in
alfalfa (Graham 1992).

For forest tree species, studies are underway of protein synthesis and
gene expression during mycorrhizal infection in Eucalyptus (Martin et al.
1992, Tagu et al. 1992), and of the regulation of gene expression during
symbiosis in Casuarina (Fana et al. 1992). Recombinant DNA techniques
are providing a valuable tool for the study of these processes, but
commercially useful manipulation by genetic engineering remains a distant
prospect.

Plants have a variety of mechanisms for coping with water stress,
generally under the control of multiple genes. A common approach to their
study has been the isolation of proteins expressed specifically during water
stress, and the isolation and sequencing of corresponding cDNA clones
(Mullet et al. 1992). Some apparent homology of dessication related genes
among distantly related plants has been demonstrated (Iturriaga et al.
1992, Cairney et al. 1992). cDNAs encoding dessication proteins in the
resurrection plant Craterostigma plantagineum were used for
Agrobacterium mediated transformation of tobacco, but transgenic plants
expressing these proteins displayed no phenotypic or growth differences,
and no improvement in dessication tolerance (Iturriaga et al. 1992). In
another study, cDNA clones of water-deficit genes from the arid woody
shrub Atriplex canescens were isolated, and transient expression achieved
in transgenic pine tissues (Cairney et al. 1992). The expression of betaine
aldehyde dehydrogenase, catalysing the last step in the synthesis of
betaine, accumulated during salt or water stress, has been under study in
sugar beet (Hanson 1990).

Genes encoding heat shock proteins, which perhaps act as “molecular
chaperones” in preventing or repairing cellular damage, have been clones
for pea and Arabidopsis (Helm et al. 1992). In this work, antisense
transformation of Arabidopsis to reduce the level of HSP21 by 50%
resulted in no phenotypic changes. Transformants overexpressing HSP21
showed detrimental effects - greatly reduced size and early flowering,
illustrating that manipulation of HSP expression with the goal of increasing
thermotolerance is a complex problem.

Specific expression of genes, e.g. CuZn-superoxide dismutase (SOD),
in response to exposure to sulphur dioxide, oxides of nitrogen (Karpinski
et al. 1992), and ozone (Wegener-Strake et al. 1992) has been under study
for some tree species.

Recombinant DNA technology is thus providing a valuable new tool
for the study of the molecular control of these and other complex traits,
but commercially important manipulation through genetic engineering
remains a distant prospect. Existing technology does not permit the ready
manipulation of complex pathways (De Waele 1992).

Species for which transformed plants have been produced were listed
recently by Birch (1993):

Table 2. Species for which transgenic plants reported (Birch 1993)

Year

Crops

Vegetables

Ornamental/ Medicinal

Fruit/ Trees

Pastures

1985

tobacco, petunia

1986

tomato, cabbage, cucumber

lotus

lucerne

1987

sunflower, Brassica spp., cotton, flax/linseed

lettuce, carrot

Arabidopsis

popular

white clover

1988

soybean, maize, rice, moth bean

potato, celery, cauliflower, asparagus, eggplant

walnut

Styosanthes, orchard grass

1989

sugarbeet

broccoli

kalanchoe

apple, Azadirachta

1990

kale, mustard

pea, muskmelon, capsicum

geranium, licorice, duboisia

Lisianthus, foxglove

tamarillo, strawberry, grape, citrus, papaya

1991

Vicia

chrysanthemum, carnation, rose, datura

pepino, pear, kiwifruit

1992

sugarcane, wheat, oats, oilseed rape, safflower

Phaseolus, tomatillo, sweet potato

Dendroboim, belladonna, poppy

craneberry, spruce, apricot

fescue, medic

1993

barley, peanut

At least 400 field trials have been established to 1993, involving genes
to improve insect resistance, viral and fungal resistance, weed control,
product flavour or other post-harvest qualities. It has been predicted that
the first products from transgenic plants are likely to be widely available
to consumers in 3–5 years (Birch 1993).

While it is true that tropical crops such as cassava, sweet potato, yam,
pulses, banana and plantain have received less attention than others (van
Montagu 1992b), tropical crops are nevertheless now the subject of quite
active research programmes aimed at genetic engineering of important
traits, for example:

An important question is the extent to which the insertion of novel or
modified genes disrupts other processes. It has been suggested that stress
resistance requires energy (Tal 1983), and that fitness costs involved in
the use of resistance genes will play an important role in decisions
concerning their use (Burdon & Jarosz 1989). Such costs may vary greatly
- low for B.t. toxins, effective at low concentrations, but perhaps higher
for genes such as proteinase inhibitors, which depend on high levels of
expression for effectiveness (Strauss et al. 1991). Glyphosate or
phosphinothricin resistant mutants have been reported to display lower
fitness than wild-type plants, the mutation in the active centre amino acids
conferring resistance to the inhibitor perhaps also resulting in a loss of
catalytic properties (Schulz et al. 1990). Few experimental data are
available for transgenic plants at this stage. Virus resistance engineered by
the coat protein approach has resulted in satisfactory protection under field
conditions, without reduction of yield (Hoekema et al. 1989, Harms 1992).
At the end of the second growing season, poplar clones transformed with
constructs containing the potato proteinase inhibitor gene, on the other
hand, displayed significantly lower diameter than untransformed plants of
the same clones (McNabb et al. 1991). In some cases, new genes may
have unpredictable effects on metabolism - for example expression of the
full length B.t. gene was toxic to tobacco cells (Strauss et al. 1991). This
discussion underscores the importance of thorough field testing prior to
release of transgenic varieties.

A series of generic patents on gene manipulation technologies may
develop as a major hurdle to commercial application of transformation - patents are held on Agrobacterium vectors, particle bombardment, the 35S
promoter, and antisense technology at least (Birch 1993).

An important selection criterion for many agricultural crops, insect
tolerance has been of low priority in breeding programmes with most
industrial forest plantation species. Some attention has been given to this
trait in poplar programmes, where volume losses (at 5 years) resulting from
insect attack have been estimated at 18% (Solomon 1985). Insect
susceptibility certainly has been a factor limiting interest in some Meliaceae
as commercial species. Conceivably, genetically engineered resistance may
be of value also with some species for which a certain level of insect
damage is currently tolerated or ignored. Attack by the Nantucket pine tip
moth (Rhyacionia frustrana), for example, has been reported to result in
significant reduction in volume in loblolly and other southern pines (Hedden
& Hangen 1987). Among non-industrial species, insect resistance is
important in Leucaena leucocephala, but generation intervals are short,
resistance is available in related species, and hybridisation offers a simple
means of introducing resistance.

Minimisation of insect damage through genetic resistance, rather than
insecticide application, is consistent with the concept of low input,
sustainable, environmentally responsible agriculture (Strauss et al. 1991,
Harms 1992). Genetic engineering of insect resistance into forest tree
species, however, is potentially more challenging than for annual crops,
where genotypes can be replaced with new ones as resistance falters.
Insertion of an array of different resistance genes, e.g. proteinase inhibitors
and B.t. genes, may be required to ensure stability of resistance for the
rotation.

The incidence of viral diseases is one of the most important factors
limiting productivity in agricultural crops, and development of resistance is
therefore a major objective of many breeding programmes. By contrast, no
forest tree improvement program includes virus resistance as a selection
criterion. Viruses have been held responsible for growth losses of 30–40%
in poplars (Cooper 1992), have been reported in some other hardwoods,
but are largely unknown in conifers (Strauss et al. 1991).

Fungal diseases cause significant growth losses in many major forest
plantation trees, e.g.: poplars, where a 35% reduction of the growing
season in clones sensitive to Melampsora rust has been reported (Ontario
Tree Imp. and Forest Biomass Institute Forest Res. 1987); slash and
loblolly pines, for which a volume reduction of 18% can result from 50%
infection (Harrison & Pienaar 1987); and radiata pine. Resistance to fungal
disease is a significant selection criterion in breeding programmes for these
species, especially the poplars.

Many tree species are quite sensitive to weed competition, and adequate
control is essential. Plantation silvicultural systems generally aim at
allowing the tree crop to establish control of the site as soon as possible.
Commonly this involves some use of herbicides in the early period
following planting. Such applications are minimized due to cost and
environmental factors. The availability of herbicide resistant plants would
permit “over the top”, as opposed to guarded, applications. This is unlikely
to be a major economic benefit for species where only one or two
applications are involved. Furthermore, environmental legislation in some
countries may impede any moves likely to increase the use of herbicides
in forested areas. Apart from environmental problems, overuse of
herbicides may result in the development of resistance in weed species, as
reported for S-triazine herbicides (Hughes 1983). It should be noted,
though, that genetic engineering of herbicide tolerance might permit some
useful substitution of herbicides - e.g. of the environmentally “friendly”
glyphosate for residual herbicides currently applied prior to planting in some
programmes. This may be a very important factor in some locations.

Many industrial plantation species are grown as exotics. Due to
desirability of silvicultural features, wood properties etc, many are being
grown in environments which, climatically, are not matched completely
with the natural range. In some cases, adaptation to the new environments
has been marred only by poor tolerance to occasional very low
temperatures. Examples are the losses of large areas of eucalypts in Florida
and southern France during the severe freezes of the mid-1980s. At the
provenance level, poor cold tolerance of southern provenance conifers in
northern Nordic areas is another example. In the first example, successful
use of eucalypts in these areas would require tolerance to temperatures
several degrees lower than those to which the species are naturally
tolerant. This would be a much larger shift than that reported in preliminary
work with cold tolerance genes discussed above.

Several components of wood quality are characterised by high
heritability, and wood quality (in particular density) is a selection criterion
in many breeding programmes. In many, selection is applied not intensely,
but rather in the form of culling of particularly undesirable genotypes. The
removal of lignin is an expensive component of pulp production, both
economically and in environmental terms, and even modest reductions in
the lignin content of wood would be of great value for pulping species such
as Eucalyptus grandis. Traditional breeding programmes with these species
do not include lignin reduction as an objective, perhaps owing to
insufficient variation within species. As noted above though, early results
offer hope that significant reductions may be achievable through genetic
engineering. Suggestions that 10–15% reductions in lignin content will not
result in major structural deficiencies in the plant are supported by work
with inhibitors and rubbery wood disease in apple (Whetton & Sederoff
1991, Dean & Eriksson 1992). Nevertheless, proper field testing of
structural properties, disease and insect resistance of lignin-reduced trees
will be essential prior to deployment. The reduction of lignin is not a useful
goal in plantations grown to provide wood for structural purposes or for
fuel.

Male sterility is used in many crop species, mainly for the commercial
production of hybrids. While male sterility would be of value for the
production of some hybrids with forest tree species, other viable
approaches to hybrid production are generally available, and this would be
a minor applications. More important potential benefits of sterility in forest
tree species are:

Increased vegetative growth, resulting from redirection of resources
otherwise committed to reproductive development. A review by
Strauss (1992, unpublished) suggests potential gains of at least 16%
in radiata pine and Douglas fir, and perhaps even higher for some other
species.

Prevention of the escape of genes into wild populations. Many
plantation programmes involve species which are not exotics, and
strategies to prevent the release of novel genes engineered into
plantation trees will be important. Such release, for example, might
accelerate counter evolution by insects to engineered resistance genes
(Strauss et al. 1991).

While effects of sterility on vegetative growth may be complex and
difficult to predict, there is no doubt that sterility will greatly facilitate
major applications of genetic engineering. In both cases, both male and
female sterility will be desirable.

For agricultural crops, the insertion of new genes into established, proven
genotypes has been the approach to integration of genetic engineering into
traditional improvement programmes most commonly applied or envisaged.
For forest tree species, alternatives which can be considered are:

The engineering of new genes into commercially proven clones. These
new genotypes are then field tested for stable integration and absence
of any pleiotropic effects, and then go into the commercial propagation
program. This is essentially the agricultural crop approach. The breeding
program underlying a clonal forestry operation such as this could
comprise:

Recurrent cycles of selection, crossing and progeny trials (in which
parents of the next generation are identified).

In a system such as this, the clonal testing program draws on the best
products of the recurrent crossing program, but commercial clones do
not re-enter the crossing program (or at least they do not need to). This
would be compatible with the requirement for sterility in engineered
genotypes. There would be no requirement to re-engineer clones to
restore fertility for continued use in breeding. Nevertheless, there are
some disadvantages associated with the approach:

Some of the tested superior clones will not be amenable to the
transformation and regeneration procedures. There is thus a selection
penalty.

There are two consecutive testing phases before clones are available
commercially - the clonal test prior to transformation, and then testing
to confirm appropriate expression in the transgenic plants. There is thus
also a time penalty involved.

The engineering of new genes into juvenile clones prior to testing. This
is similar to the above, except that the novel genes are introduced to
clones prior to testing. Juvenile material, e.g. immature embryos, from
the families selected as donors of genotypes for clonal testing is
subjected to the transformation procedures. A marker could be used to
select for transgenic plants. The transgenic plants are then established
in a clonal test, which serves to identify genotypes which are desirable
with respect to both traditional traits and the expression of the novel
genes. This avoids the disadvantages above - resources are not wasted
on clonal testing of genotypes not amenable to transformation and
regeneration, and clonal testing and testing for expression of novel
traits are conducted concurrently. An additional advantage is that
transformation and regeneration are conducted with juvenile tissues,
usually much more amenable. A larger number of genotypes must be
transformed - but this need not be a problem. It is essential though that
the absence of adverse genetic correlations between major commercial
traits and competence for transformation and regeneration be
confirmed.

Transformation of known good parents, and then use of these in seed
orchards. Involving the simple addition of a genetic engineering step to
traditional seed orchard technology, this approach is superficially
attractive. The same disadvantages outlined for option 1 above apply
- some selections will not be amenable to the procedures, and there will
be a time penalty for confirmation of appropriate expression. Other
disadvantages will be:

The approach is not compatible with the principle of prevention of
escape of genes through use of sterility. This is likely to constitute a
major obstacle to the integration of genetic engineering into seed
orchard programmes.

Not all seed orchard progeny will carry the novel genes, even in the
absence of pollen contamination and for completely dominant genes.

Transformation of microspores. Microspores of Brassica napus have
been transformed by electroporation (Jardinaud 1992). For tobacco, an
in vitro system that allows effective maturation of microspores into
pollen grains that can be used to pollinate emasculated flowers in situ
has been developed (Alwen et al. 1990). Nevertheless, many technical
difficulties remain to be overcome with this approach. Transgenic
pollen grains could be used in either of two ways:

In the breeding program. Fertility of progeny would be a requirement,
leading to the obstacle discussed above regarding escape of genes into
wild populations.

For pollinations to yield genotypes for clonal testing (forward selected
families as described above). Fertility of the progeny would not be
required, and sterility genes could be included in the constructs.

At this stage, it seems likely that the clonal test will be the most
appropriate basis for the integration of genetic engineering into breeding
programmes. Transformation is probably most efficiently incorporated prior
to clonal testing, and transformation of mature material is therefore not
essential.

Work with the genetic engineering of forest tree species is advancing
rapidly, and there are likely to be many more reports of transformation of
forest tree species with marker genes and the simple genes such as B.t.
and glyphosate resistance over next couple of years.

The availability of effective transformation techniques remains an
obstacle, but improved techniques are being developed. Regeneration is a
difficulty for some tree species, but the problem may be over - rated - the
non-competence of mature material is not necessarily an obstacle to
effective application of genetic engineering, provided that juvenile material
responds satisfactorily. Major traits for which genetic engineering can most
realistically be contemplated in the near future include virus resistance,
insect resistance and herbicide tolerance. Even so, insertion of one of these
genes into a new species would be a substantial undertaking, and insertion
of enough genes to confer long term insect resistance in a perennial
species (particularly for long rotations) more so. Virus and insect
resistance, in particular, are of major significance for crop plants. By
contrast, these traits are not among the most important for most forest
tree species. Reduction of lignin is a valuable objective for pulp species,
and prospects look good. Cold tolerance is a trait of considerable interest,
particularly in some eucalypt species. Much remains to be done, though,
to establish that sufficient tolerance can be conferred using antifreeze
proteins, and to extend the work to tree species. Prevention of the escape
of genes into wild populations is likely to become an important concern,
and sterility should be an early target of genetic engineering work with
forest tree species. The major factor limiting application of genetic
engineering in forest tree species is the state of knowledge of molecular
control of the traits which are of most interest - those relating to growth,
adaptation and stem and wood quality. Genetic engineering of these traits
remains a distant prospect. An often overlooked research component is the
testing which would be required before a responsible recommendation for
large scale deployment of transgenic plants could be made. Such testing
could be extensive and prolonged, depending on the species and genes
involved.

It is important that genetically engineered genotypes be of high quality
with respect to other traits as well. The clonal test is the most logical basis
for integration of genetic engineering into traditional tree improvement
programmes. For these reasons, genetic engineering is most appropriately
conducted with species where breeding programmes are advanced and
clonal forestry can be realistically contemplated. Genetic engineering
represents, for many crop plants, the best hope for addressing the major
priorities of breeding programmes - the acquisition of virus and insect
resistance. This applies also for some crops in developing countries, e.g.
cassava. By contrast, genetic engineering can do little, at the present time,
to address the major priorities of breeding programmes for non-industrial
tree species in developing countries.